Thymidylate synthase (TS) is an important target for chemotherapy drugs such as 5-fluorouracil and raltitrexed. Over-expression of TS has been linked to chemotherapy resistance. A polymorphic tandem repeat sequence in the 5' untranslated region (5'UTR) of the human TS gene (TSER) has been shown to influence TS expression. The presence of a triple tandem repeat (TSER*3) increases in vitro TS expression compared to a double tandem repeat (TSER*2) and is associated with higher in vivo tumor TS activity. The polymorphism of this promoter enhancer region has not been extensively studied in patients with cancer and may represent a possible mechanism of intrinsic resistance to TS inhibitors. In this study, PCR analysis of genomic DNA from 121 patients with colorectal cancer demonstrated 29% of patients were homozygous for TSER*3, 16% were homozygous for TSER*2 and 55% were heterozygous. In 44/45 microdissected tumors the TS enhancer genotype was identical between paired samples of colorectal tumor and normal tissue. In 24 patients receiving a bolus/infusion 5-fluorouracil (5FU) regimen for metastatic colorectal cancer, 22% of non-responders to chemotherapy were homozygous for TSER*2 compared with 40% of responders. Median survival dropped from 16 months for homozygous TSER*2 to 12 months for homozygous TSER*3. This is consistent with previous studies where higher TS expression was associated with poor response to TS inhibitors. Prospective analysis of the influence of the TS polymorphism on patient outcome is warranted.